Preparation of Frozen Stock (C2010)

Introduction

Culture should be approximately 90% confluent prior to preparation of frozen stock in order to insure the highest number of viable cells from reviving a frozen culture.

Protocol

1. Transferred one-day old growing culture into a sterile 50 ml conical tube.
2. Centrifuge culture at 1,500 rpm for 2-3 minutes at room temperature. Centrifugation at > 1500 rpm will result in cell death.
3. Remove and discard supernatant by gently pipetting it out without disturbing the cell pellet.
4. Resuspend cell pellet in 10 ml of RPMI-1640 with 10% FBS. Repeat centrifugation and remove and discard supernatant by gently pipetting it out without disturbing the cell pellet.
5. Add the appropriate volume of Cryoprotectant Medium (RPMI-1640 containing 50% FBS and 10% DMSO) to the cell pellet to achive a concentration of at least 2 x 10⁶ cells per ml.
6. Resuspend pellet by gently pipetting up and down 2-3 times.
7. Aliquot 0.5-1 ml of cell suspension into cryogenic tubes.
8. Place tubes in an isopropanol-filled cryostorage container (be sure the tubes are capped tightly).
9. Transfer the cryostorage containter to -70°C overnight.
10. The next day transfer frozen cells immediately to liquid nitrogen vapor phase storage.
    
    Note: After an overnight at -70°C, a vial of frozen cells should be revived and tested to ensure that the frozen cells are still viable and functional.